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| Status |
Public on May 13, 2016 |
| Title |
H3K36me2_ChIPseq H3.3K36M |
| Sample type |
SRA |
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| Source name |
C3H10T1/2 cells & S2 cells
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| Organism |
Mus musculus |
| Characteristics |
cell line: C3H10T1/2
cell type: C3H embryo-derived mesenchymal progenitor cells
genotype: Stably expressing FLAG-HA-tagged K36M mutant H3.3
passages: Passage 10-15
spike-in: soluble chromatin from Drosophila melanogaster S2 cells derived from a primary culture of 20–24 hours old, late stage embryos (equivalent to 5% of the mouse cell chromatin)
chip antibody: H3K36me2 antibody (Millipore, catalog # 07-369-I, lot # 2475921)
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| Growth protocol |
C3H10T1/2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) with 10% fetal bovine serum (FBS, CellGro). Drosophila S2 cells were cultured in Schneiderís Drosophila Medium (Invitrogen) containing 10% heat-inactivated FBS (CellGro).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
~2x10^7 C3H10T1/2 cells were lysed using digesting buffer (50 mM Tris-HCl pH 7.6, 1 mM CaCl2 and 0.2% Triton X-100) and digested with micrococcal nuclease to obtain mono-nucleosomes. The chromatin were then dialyzed into RIPA buffer (10 mM Tris pH 7.6, 1 mM EDTA, 0.1% SDS, 0.1% Na-Deoxycholate, 1% Triton X-100) for 2 h at 4°C. After centrifugation, soluble chromatin was spiked-in with soluble chromatin from Drosophila S2 cells that was similarly prepared and equivalent to 5% of the mouse cell chromatin. The mixed soluble chromatin was incubated with αH3K36me3 (Active Motif, 61101), αH3K36me2 (Millipore, 07-369-I) or αH3K27me3 (Cell Signaling Tech, 9733) antibody bound to 75 μl protein A or protein G Dynal magnetic beads (Invitrogen) and incubated overnight at 4°C, with 5% kept as input DNA. Magnetic beads were washed with RIPA buffer and LiCl buffer (0.25 M LiCl, 0.5% NP40, 0.5% Na-Deoxycholate) and chromatin was eluted. ChIP DNA was treated with Proteinase K (Roche) and recovered using Qiagen PCR purification kit.
libraries were prepared according to the Illumina TruSeq protocol
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
ChIP-DNA
Peaks_K36me2_RxNormalizedReads
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| Data processing |
Reads that passed the quality score were aligned to mm9_dm6 custom genome using bowtie 1.0.0 with default parameters. The mm9_dm6 custom genome was built by concatenating the mm9 and dm6 genomes.
Sample normalization factor (Rx) was determined as 1 over the number of reads mapping to Drosophila genome per million
Peaks were called using mosaics Input-only, Two-signal analysis. Background was calculated using robust method of moment. (threshold = 20, FDR= 0.05, maxgap = 10000bp, minsize = 5000bp, binSize= 200bp, two component model, Input-Only analysis)
Reads belonging to peaks were determined for H3.3WT and H3.3K36M samples and normalized by the Rx-normalization factor
Genome_build: mm9/dm6
Supplementary_files_format_and_content: tab-separated file containing genomic coordinates of H3K27me3, H3K36me2 and H3K36me3 peaks in H3.3WT and H3.3K36M cells
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| Submission date |
May 27, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Peter W Lewis |
| E-mail(s) |
pwlewis2@wisc.edu
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| Phone |
608-316-4388
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| Organization name |
University of Wisconsin-Madison
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| Department |
Department of Biomolecular Chemistry
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| Lab |
Room 2174, Wisconsin Institute for Discovery
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| Street address |
330 N Orchard Street
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| City |
Madison |
| State/province |
Wisconsin |
| ZIP/Postal code |
53715 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE69289 |
H3K36 mutations promote sarcomagenesis through genome-wide remodeling of H3K36 and H3K27 methylation [ChIP-Rx] |
| GSE69291 |
H3K36 mutations promote sarcomagenesis through genome-wide remodeling of H3K36 and H3K27 methylation |
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| Relations |
| BioSample |
SAMN03737786 |
| SRA |
SRX1040843 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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